Enzymes make up the largest class of naturally occurring proteins. One class of enzyme includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has been exploited by incorporating naturally occurring and protein engineered proteases into cleaning compositions, particularly those relevant to laundry applications.
In the cleaning arts, the mostly widely utilized of these proteases are the serine proteases. Most of these serine proteases are produced by bacterial organisms while some are produced by other organisms, such as fungi. See Siezen, Roland J. et al., “Homology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteases”, Protein Engineering, Vol. 4, No. 7, pp. 719-737 (1991). Unfortunately, the efficacy of the wild-type proteases in their natural environment frequently does not translate into the unnatural cleaning composition environment. Specifically, protease characteristics such as, for example, thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.
Several approaches have been employed to alter the wild-type amino acid sequence of serine proteases with the goal of increasing the efficacy of the protease in the unnatural wash environment. These approaches include the genetic redesign of proteases to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
However, because such genetically engineered proteases are foreign to mammals, they are potential antigens. As antigens, these proteases cause immunological and allergic responses (herein collectively described as immunological responses) in mammals. In fact, sensitization to serine proteases has been observed in environments wherein humans are regularly exposed to the proteases. Such environments include manufacturing facilities, where employees are exposed to the proteases through such vehicles as uncontrolled dust or aerosolization. Aerosolization can result by the introduction of the protease into the lung, which is the route of protease exposure which causes the most dangerous response. Protease sensitization can also occur in the marketplace, where consumers' repeated use of products containing proteases may cause an allergic reaction.
Furthermore, while genetic engineering has been prominent in the continuing search for more highly effective proteases for use in laundry applications, genetically engineered proteases have been minimally utilized in personal care compositions and light duty detergents. A primary reason for the absence of engineered proteases in products such as, for example, soaps, gels, body washes, and shampoos, is due to the aforementioned problem of human sensitization leading to undesirable immunological responses. It would therefore be highly advantageous to provide a personal care composition which provides the cleansing properties of engineered proteases with minimized provocation of immunological responses.
One approach toward alleviating the immunological activity of a protease is through the redesign of one or more epitopes of the protease. Epitopes are those amino acid regions of an antigen which evoke an immunological response through the binding of antibodies or the presentation of processed antigens to T cells via a major histocompatibility complex protein (MHC). Changes in the epitopes can affect their efficiency as an antigen. See Walsh, B. J. and M. E. H. Howden, “A Method for the Detection of IgE Binding Sequences of Allergens Based on a Modification of Epitope Mapping”, Journal of Immunological Methods, Vol. 121, pp. 275-280 (1989).
The present inventors have discovered that those serine proteases commonly known as subtilisins, including subtilisin BPN′, have a prominent epitope region at amino acid positions 70-84 corresponding to BPN′. The present inventors have herein genetically redesigned such subtilisins to alleviate the immunogenic properties attributed to this epitope region. In so doing, the present inventors have discovered subtilisins which evoke a decreased immunological response yet maintain their activity as an efficient cleansing protease. Accordingly, the present proteases are suitable for use in several types of compositions including, but not limited to, laundry, dish, hard surface, skin care, hair care, beauty care, oral, and contact lens compositions.